Today most biopharmaceuticals are produced in animal (mammalian) cells, which usually secrete the recombinant polypeptide of interest with high efficiency, quality and appropriate post-translational (secondary) modifications (such as e.g. glycosylation) into the cultivation medium. However, some fusion polypeptides, especially complex fusion polypeptides, polypeptides with low solubility or folding difficulties, as well as polypeptides interacting with the cell expressing it are often obtained at very low yields.
For example, antibodies are usually expressed in mammalian cells with high efficiency in biologically active form. However, fusion polypeptides comprising an antibody, e.g. green fluorescent protein (GFP) antibody conjugates, are expressed and/or secreted not at all, although such proteins would be highly interesting for experimental and diagnostic approaches (see e.g. WO 2011/135040).
In certain cases difficult to express polypeptides can be produced as soluble secreted inactive precursor proteins e.g. so called zymogens in the case of proteases which can be matured afterwards in vitro e.g. by proteolytic activation. In other cases polypeptides, which are detrimental for a specific host cell, can be expressed as inactive insoluble protein aggregates within the cell (inclusion bodies (IBs)) and afterwards refolded in vitro. However the processing of pro-polypeptides can be difficult or not possible at all. Moreover, the obtained mature polypeptides do not contain all posttranslational modifications.
Examples of poorly expressed polypeptides are neurotrophic factors like NGF, BDNF, GDNF and NT-3 (see e.g. Xia, Ch.-F., J. Gene Med. 10 (2008) 306-315; Boado, R. J., Pharm. Res. 24 (2007) 1772-1787; Negro, A., et al., J. Neurochem. 62 (1994) 471-478).
In WO 2008/005847 a method of producing factor viii proteins by recombinant methods is reported. Peptide extended glycosylated polypeptides are reported in WO 02/02597. In WO 2007/044323 fusion proteins for blood-brain barrier delivery are reported. Nerve cell growth modulators (amphibodies) are reported in WO 00/64482. In WO 2012/087835 are reported compositions and methods for enhancing protein folding. Stable and soluble antibodies inhibiting TNFalpha are reported in WO 2006/131013. In WO 00/23473 interferon-beta fusion proteins and uses are reported.